N5(carboxyethyl)ornithine synthase (CEOS), an enzyme discovered in Lactococcus lactis, mediates the NADP-dependent condensation of ornithine (or lysine) and pyruvate to yield N5(carboxyethyl)ornithine, (or N6(carboxyethyl)lysine). To examine the structure and function of this enzyme in more detail and to analyze its relationship to other N(carboxyalkyl)amino acid dehydrogenases, the gene ceo (which encodes CEOS) was cloned in Escherichia coli and sequenced. An open reading frame of 313 amino acids with a deduced molecular weight of 35,323 was detected. The first 37 amino acids of the sequence thus obtained agreed with the results from the stepwise Edman degradation of purified CEOS. A putative ribosome binding site (5'-AAGGA) was detected 8 nucleotides upstream from the translation initiation codon. Further analysis indicated that: 1) CEOS is not closely related (> 30% identity) to any protein in the data banks; 2) the aligned sequences of CEOS and nopaline dehydrogenase from Agrobacterium tumefaciens showed 21.7% identity; 3) the aligned sequences of CEOS and octopine dehydrogenase from A. tumefaciens showed 18.5% identity; 4) CEOS contains an NADP-binding motif (GXGXXA); 5) CEOS also contains the sequence DXXR which may be involved in the binding of pyruvate and catalysis. In L. lactis K1, ceo is located on a large transposon (Tn5306) which also encodes the abilities to produce the small protein antibiotic nisin and to ferment sucrose. Hybridization studies with oligonucleotide probes for the genes encoding EII Scr (scrA), sucrose-6-phosphate hydrolase (scrB) and fructokinase (scrK) showed that scrA, scrB, and scrK are closely linked and that they are located on the same 20-kb EcoRI fragment that encodes ceo (order ceo-scrKAB). The nisin precursor gene spaN is located on a 14-kb EcoRI fragment. In L. lactis ATCC 11454, spaN, scrA, scrB, and scrK (but not ceo) are located on a related transposon, Tn5307.